»D £U9 

.S7 B8 
1922 
Copy 1 



EFFECT OF DIFFERENT REACTIONS ON THE GROWTH AND 
NODULE FORMATION OF SOYBEANS 



BY 
O. C. BRYAN 



PH. D. THESIS 



Wisconsin Agricultural Experiment Station 



Reprinted from 



Soil Science, Vol. XIII, No. 4, April, 1922 



Reprinted from Soil Science 
Vol. XIII, No. 4, April, 1922 






vp 



EFFECT OF DIFFERENT REACTIONS ON THE GROWTH AND 
NODULE FORMATION OF SOYBEANS 1 

O. C. BRYAN 

Wisconsin Agricultural Experiment Station 
Received for publication July 20, 1921 

Although it is well known that soil acidity and alkalinity have a marked 
effect on cultivated plants, especially certain legumes, yet, it has never been 
definitely determined at what reaction the legumes grow best and become 
inoculated most abundantly. Whether the favorable range of reaction is wide 
or narrow, and what this range is for each of the legumes are very important 
questions from both a practical and a scientific standpoint. In order to gain 
further information on these questions, the present investigation was under- 
taken. 

The soybean was used in the beginning because of its adaptation to water 
cultures and because of its importance as a crop. Several different strains of 
soybean bacteria were studied. For comparison, some results with corn and 
cowpeas obtained under similar conditions are reported herein. 

An exhaustive discussion of the literature on this subject does not seem 
necessary and only a brief review of some of the more important papers is 
given. The magnitude of the hydrogen- and hydroxy 1- ion concentration of a 
number of soil solutions and suspensions have recently been determined by 
Gillespie (15), Sharp and Hoagland (35), Plummer (32), Truog (37), and 
others. These investigations have shown that soil solutions have a wide 
range of reactions, namely, from pH 3 to pH 9. 

The importance of the effect of the soil reaction on the soil bacteria is well 
recognized. Gruzit (14) reported that an alkaline reaction was more favorable 
for the growth of the bacteria than an acid, or even neutral reaction. Salter 
(33) reported that red clover bacteria do best at a neutral or slightly acid reac- 
tion while the alfalfa bacteria do best at a slightly alkaline reaction. Fred 
and Loomis (11) reported that an alkaline reaction (pH 7.72) produced maxi- 
mum growth of the alfalfa bacteria. Fred and Davenport (12) reported that a 
correlation exists between the acid resistance of the nodule bacteria and the 

1 Part I of a thesis submitted at the University of Wisconsin in partial fulfillment of the 
requirements for the degree of Doctor of Philosophy. 

Published with the permission of the Director of the Wisconsin Agricultural Experiment 
Station. . 

The writer wishes to express his appreciation for the helpful suggestions and criticisms 
tendered by Professors E. Truog and E. B. Fred under whose direction the work has been 
done. a 

271 



272 O. C. BRYAN 

acid resistance of the higher plants. Bewley and Hutchinson (2) reported 
that some of the legume bacteria are either killed in definitely acid soils, or at 
least lose their activity, while Hiltner (21) claimed that liming has an in- 
jurious effect on lupine bacteria. 

The sensitiveness of plants to the reaction of dilute solutions of acids,bases 
and salts was noted by Kahlenberg and True (26), Heald (20) and Loew (29). 
These investigators studied primarily the direct chemical effect of reaction on 
seedlings and not the influence on growth, and hence did not use balanced 
nutrient solutions. Because of this condition definite conclusions can not be 
drawn from their data regarding influence on growth. Their data show, how- 
ever, that seedlings differ widely in their behavior toward reaction. 

Cameron and Breazeale (4) reported that corn was much more acid tolerant 
than wheat or clover. Hartwell and Pember (18) claimed that some of the 
cereals are more sensitive to acidity than others but no statement was made 
as to the relative sensitiveness. Hoagland (23) reported that barley seedlings 
grew better in a slighty acid reaction than in a neutral or alkaline reaction. 
This was also reported by Salter and Mcllvane (34) in working with corn, 
wheat, soybeans and alfalfa. The data of the latter indicate that alfalfa is 
more sensitive to acidity than corn, wheat or soybeans. Joffe (25) reported 
that alfalfa produced good growth in soil cultures which were made very acid 
(pH 3.8) by the addition of sulfuric acid. Hixon (22) noted that there was a 
difference in water content, organic matter, and total ash in the roots of 
16-day-old wheat seedlings grown at different reactions. 

Hartwell and Pember (18) and Dachnowski (10) claimed that the hydrogen 
ion was more toxic to plant growth than the hydroxyl ion. This question was 
investigated by Hoagland (23) who reported the opposite results with barley 
seedlings. These contradictory results are possibly due to different methods 
of determining the reactions. Whether total acidity or hydrogen ion con- 
centration is used as a criterion for indicating reactions is of great importance. 
Other factors, e. g., kind of nutrient solution used and proper maintenance of 
the desired reaction, are also important. 

The change in reaction of the nutrient solution in contact with growing plant 
roots was noticed by Hartwell and Pember (18) who reported that an acid 
solution tended to become alkaline. This was also reported by Breazeale and 
LeClerc (3) who claimed from their work with wheat seedlings that the change 
in reaction of the nutrient solution was due to the selective absorption of the 
ions by the plants. The buffer condition of the nutrient solution and the fre- 
quency of renewing the solution are no doubt important factors in maintaining 
a constant reaction. The change in reaction of the nutrient solution in 
contact with plant roots during 1-day periods was found by Hoagland (23) 
to be quite considerable provided the initial reaction was not favorable. 

Salter and Mcllvane's (34) results indicate that their nutrient solution 
remained fairly constant over a period of 4 days when in contact with grow- 
ing plants. Duggar (9) concluded that, in general, an acid or an alkaline 






EFFECT OF REACTION ON NODULE FORMATION 273 

solution tended toward neutrality when in contact with growing plants. 
However, this change depended on the nutrient solution used and the rate of 
plant growth. The most favorable reaction also varied with the • kind of 
nutrient solution used. 

In connection with the influence of reaction on plant growth, it is well to 
mention the toxic effects of soluble aluminum salts as reported by several 
investigators. Abbott, Conner and Smalley (1) reported that the toxic 
effect of aluminum nitrate was about the same as a solution of nitric acid of 
equal normality; and that water extracts from acid soils containing soluble 
aluminum salts were as toxic to plant growth as a nutrient solution containing 
an equal amount of aluminum salts. Hartwell and Pember (19) found that 
aluminum salts were considerably more toxic to barley seedlings than to rye 
seedlings. The toxic effect of acidity alone was about the same on both 
plants. They concluded that lime may precipitate the aluminum and thus 
be of value in this way as well as in neutralizing the acidity. Mirasol (30) 
reported that aluminum salts which could be extracted from the soil with a 
solution of potassium nitrate were probably the cause of the unproductiveness 
of three acid soils of Illinois. Conner (6) reported that much of the harmful 
effects of acidity of soils is due to soluble aluminum salts; and the presence of 
abundant lime or phosphate will prevent this harmful effect. He noted that 
some plants are more sensitive to aluminum salts than others. This condi- 
tion was also noted by Hartwell. 

Undoubtedly, in very acid soils aluminum, manganese and other toxic sub- 
stances go into solution and produce injurious effects on cultivated plants. 

The results of the various investigations may be summarized as follows: 
The range of reaction of different soils is sufficiently wide to give conditions of 
acidity and alkalinity in some cases which are unfavorable to bacteria and 
higher plants. The different legume bacteria vary in their behavior toward 
reaction, and the degree of the acid resistance appears to be in the same 
direction as that of the host plants. In the case of plants, there are some 
contradictory results, but, in general, they indicate that plants differ in their 
behavior toward reaction. This is in line with field observations. In studying 
a problem of this nature, there are a number of factors which are difficult 
to control and the conflicting results obtained by the different investigators 
are perhaps due in part to the difficulty of controlling the reactions, and in 
some cases to the use of unfavorable nutrient solutions. 

In regard to the toxic effects of soluble aluminum and other substances in 
acid soils, it appears that plants vary in their behavior toward these toxic 
substances and that the harmful effects of acidity in some soils are due in 
part to soluble aluminum salts. 

GROWTH OF DIFFERENT STRAINS OF SOYBEAN BACTERIA AT VARIED REACTIONS 

, Since some investigators (2) had noted that many of the legume bacteria are 
either killed, or, at least rendered inactive in acid soils, it was thought best, as 



274 O. C. BRYAN 

a preliminary experiment, to determine the critical hydrogen- ion concentra- 
tion of the various strains of soybean bacteria in pure cultures and also to 
determine if there is any marked difference in behavior of these strains of 
bacteria at different reactions. By strains of bacteria, is meant a pure culture 
isolated from a known variety of soybeans. 

Twenty-one 2 strains of soybean bacteria were grown on mannit, on sucrose 
and on soil-extract agars. The mannit and sucrose agars were prepared 
according to Ashby's formula as follows: 

Mannit or sucrose 15.0 gm. 

MgS(V7H 2 0.2 gm. 

KH 2 P0 4 0.2 gm. 

NaCl 0.2 gm. 

CaSCV2H 2 ; . 1 gm. 

Agar 15.0 gm. 

Distilled water 1000.0 cc. 

The soil extract agar was prepared by diluting 100 cc. of soil extract from a 
silt loam soil to 1000 cc. and adding 15 gm. each of mannit and agar. The 
range of reaction used was from pH 3.3 to pH 10 with the sucrose agar and 
from pH 3.3 to pH 7 with the mannit and soil extract agars. The desired 
reactions were obtained by adding varied amounts of sterile sulfuric acid and 
sodium hydroxide, as the case required, before the media were allowed to 
solidify in slants. The Clark and Lub's method for colorimetric determina- 
tion of the reactions of solutions was employed. Each culture was inoculated 
with one drop of suspension containing the bacteria. Triplicates of each 
reaction were used in all cases. The cultures were incubated 15 to 20 days 
at 28°C. before final results were recorded. 

In general the mannit and soil extract agars showed a more vigorous growth 
than the sucrose agar, although the three media showed only minor differences 
in critical pH for the various strains of bacteria. There, were small differences 
in growth of bacteria between the reactions pH 7 and pH 6.5 but an increase in 
hydrogen- ion concentration from pH 6.5 to the critical concentration produced 
a gradual decrease in growth with each strain of bacteria. Eight strains of 
the bacteria were grown in the alkaline range, all of which grew at pH 10. 
The maximum growth of the eight strains grown in the alkaline range took 
place at reaction of about 7.6. Table 1 gives the critical hydrogen-ion con- 
centration for the different strains of soybean bacteria studied. By critical 
hydrogen-ion concentration is meant the reaction at which the bacteria do not 
produce any visible growth during the 15 days after inoculation. It will be 
noted that there is not a great difference in the critical hydrogen- ion concentra- 
tion for the various strains of soybean bacteria. 

2 Fourteen of the different strains of soybean bacteria studied were furnished through the 
kindness of the Bureau of Plant Industry, of the United States Department of Agriculture 
One strain was furnished through the kindness of Dr. A. L. Whiting, University of Illinois.. 



EFFECT OF REACTION ON NODULE FORMATION 



275 



TABLE 1 



The critical hydrogen-ion concentration for soybean bacteria on mannit, sucrose, and soil 

extract agar 



STRAIN OF BACTERIA 



Arlington 

Barchet 

Barchet 

Chinese 

Cloud 

Cornell 

Haberlandt , 

Haberlandt , 

Hobta 

Manchu , 

Manchu, Wisconsin Experiment Station. . . 
Medium early, Illinois Experiment Station 

Mammoth yellow 

Pingsu 

Roosevelt 

Roosevelt 

Soybean 

Taha 

Tokio 



NUMBER 


CRITICAL pH VALUE 


118 


4.2 


293 


4.2 


218 


4.6 


271 


4.6 


270 


4.2 


150 


4.5 


210 


4.0 


334 


4.0 


337 


4.0 


312 


4.7 


154 


4.6 




4.0 


338 


4.2 


275 


4.2 


• 181 


4.6 


187 


4.0 


152 


4.6 


233 


4.4 


256 


4.6 



PLANT CULTURES 



Methods 



The plants were grown in solution and sand cultures in the greenhouse from 
March 1 to July 25. The culture vessels used were 500 and 600-cc. percolators. 
These were provided with glass tubes and pinch-cocks at the bottom in order 
that the solutions might be easily removed without disturbing the plants. 
In the case of sand cultures, the percolators were supported by iron [ring 
stands and wrapped in heavy brown paper to exclude the light. Those con- 
taining solution cultures of soybeans were placed in boxes and surrounded 
with moist sawdust to exclude light and prevent rapid changes of temperature. 
The glass tubes extended through holes in the boxes in order that the solu- 
tions might be removed readily. A few solution cultures of corn and cowpeas 
were also set up. For holding these percolators, circular holes were cut of such 
size in the top of a greenhouse bench that the percolators passed through to the 
rim around the mouth which acted as a support. The light was excluded 
from beneath the bench. 

The most desirable nutrient solution for this type of investigation was un- 
known at the start. A modified form of Shive's three-salt solution was first 
used with the soybeans. The modification consisted in reducing the amount 
of Ca(N0 3 ) 2 to one-fourth that recommended by Shive, and adding an equal 
amount of CaCl«. This made a total reduction of calcium to about one-half 



276 O. C. BRYAN 

that in the regular solution. The chloride was substituted for the nitrate, 
since large quantities of nitrates are known to hinder nodule formation. The 
reduction of calcium was made to reduce the precipitation of Ca3 (P0 4 )2 in 
the alkaline solutions. This modified solution proved unfavorable for nodule 
formation of soybeans in solution cultures. Shive's regular solution with 
varied amounts of nitrates was then tried, but again no nodules developed. 
Several other nutrient solutions, namely, Pfeffer's, Hopkins-Pettit's, Crone's, 
and also Mendota Lake water were tried with the result that only in Crone's 
nutrient solution and Mendota Lake water were nodules produced. Crone's 
solution being the most satisfactory, was chosen with slight modification for 
the work reported herein. The modification will be given later. 

The reactions used in all cases were approximately pH 3, 4, 5, 6, 7, 8, 9 and 
10. For specific reactions, see table 6. The Clark and Lub's method for 
colorimetric determination of reaction of solutions was employed in making 
the adjustments. The standards were checked at intervals with the hydrogen 
electrode. In order to maintain the reaction of the nutrient solution, in 
contact with plant roots, as constant as possible the solutions were renewed 
daily. 

For the sand cultures, 20-mesh (Ottawa silica) sand was used. This was 
first thoroughly washed with distilled water. The sand was then placed in 
the percolators and washed with the respective solutions until the reaction 
remained constant on passing through the sand. The solution in sand cultures 
as in solution cultures were renewed daily. This was done by means of 
suction as sugested by McCall (31). The percolators were ideally adapted 
for changing of solutions. 

The seeds were germinated in clean quartz sand and allowed to grow 3 
to 5 days before being transferred to the percolators. Two seedlings were 
grown in each percolator both in sand and solution cultures. The seedlings, 
in case of the solution cultures, were held in place by means of paraffined corks 
and plugs of cotton. In order that the seedlings would not be subjected to a 
great change in reaction at once when being transferred to the percolators, the 
acid and alkaline reactions of the nutrient solution were brought to the desired 
points gradually over a period of 2 days. The solutions were inoculated 
3 days after the seedlings had been transferred to the percolators. This 
was done by placing the inoculum in the nutrient solutions at the time the 
solutions were renewed. The plant cultures were grown for 25 to 35 days. 

Growth of soybeans in Shive's nutrient solution at different reactions 

As previously stated, a modified form of Shive's nutrient solution was first 
used in both sand and solution cultures. It was prepared from the following 
stock solutions in which the amounts of salts are indicated on the anhydrous 
basis. 



EFFECT OF REACTION ON NODULE FORMATION 



277 



(a) CaCl 2 50.0 gm. 

Ca(N0 8 ) 2 50.0 gm. 

Distilled water 500.0 cc. 

(b) MgS0 4 50.0 gm. 

Distilled water 500.0 cc. 

(c) K2HPO4 50.0 gm. 

Distilled water 500.0 cc. 

(d) FeCU 2 . 5 gm. 

Distilled water 300 . cc. 

Ten cubic centimeters of (a), (b), (c), and ten drops of (d) were added to 
4 liters of distilled water. Dilute sulfuric acid or sodium hydroxide was added 
to portions of this solution until the desired reactions were obtained. The 
general method of procedure already outlined was followed. The plants were 
allowed to remain in the solution 24 days from time of inoculation. 

iable 2 

Growth and inoculation of soybeans in sand cultures with Skive's nutrient solution at different 

reactions 



REACTION OF CULTURE 


NODULES PER PLANT 


PLANT DEVELOPMENT 


pH 






3.2 





Tops fair 

Roots dark and stubby 


4.2 


5 


Roots better than at pH 3.2 


5.0 


12 


Tops good 

Roots slightly dark 


6.0 


35 


Tops good 
Roots good 


6.9 


49 


Tops good 
Roots good 


8.0 


11 


Tops good 

Roots slightly brown 


9.0 


2 


Tops small 
Roots very brown 


9.9 





Very poor 



The results of all plants in all experiments at different reactions will be 
referred to as at pH 3, 4, 5, 6, 7, 8, 9, and 10 although the actual pH value 
may have been slightly more or less. For specific reaction with Shive's 
nutrient solution, see table 2. 

Only two nodules developed in Shive's nutrient solution and these appeared 
at pH 8. A poor and injured condition of the plants indicated that something 
was wrong with the nutrient solution. This may have been due to the presence 
of toxic impurities in the salts used. Plants at pH 3, 4 and 10 were almost 



278 0. C. BRYAN 

dead. Sand cultures of soybeans with Shive's nutrient solution were also 
carried on according to the regular method already described. The results 
of growth and inoculation with these are given in table 2. The best growth and 
inoculation were obtained at pH 6 and 7. In contrast to plants grown in 
solution cultures, the plants grown in sand cultures showed no injurious effect 
other than that due to reaction. The plants at pH 3, 4 and 10 had very dark 
roots and small tops. In general, a considerable increase in acidity or alka- 
linity caused a decrease in plant growth. Perhaps the absence of injurious 
effects in the sand cultures was due to adsorption of toxic substances from the 
solution by the sand. 

The effect of nitrates on nodule formation in Shive's nutrient solution 

Since practically no nodules developed in solution cultures with Shive's 
solution, it was thought that possibly the presence of nitrates prohibited 
inoculation. To determine the influence of nitrates on nodule formation, 
soybeans were grown in Shive's nutrient solutions containing 2, 1, \, f , f , r&, 
3^-, and -gj times the usual amount of nitrates recommended by Shive, and 
also in one solution entirely without nitrates. 

For culture vessels, 500-cc. wide-mouthed bottles were used, and two 
soybean seedlings which had been germinated in clean quartz sand were 
placed in each. The seedlings were held in place with paraffined corks and 
plugs of cotton. The solutions were inoculated and renewed weekly. The 
plants were allowed to grow for four weeks, during which time not a single 
plant in any of the solutions developed any nodules. All the plants had 
yellowish leaves and somewhat dark and stubby roots indicating that the 
solution was toxic. 

Influence of different nutrient solutions on nodi&e formation of soybeans 

Since all attempts to get nodule formation on soybeans in solution cultures 
had practically failed, it was decided to determine the effects of different 
nutrient solutions on inoculation of soybeans. In addition to Shive's solu- 
tion, Hopkin-Pettit's, Pfeffer's and Crone's solution, and also Mendota Lake 
water were used. The cultures were carried on in exactly the same manner 
as the previous ones. At the end of 4 weeks, the plants in Crone's nutrient 
solution and Mendota Lake water had developed a goodly number of nodules 
and the plant growth was healthy. Those in Shive's nutrient solution had 
developed only two nodules and the roots were dark and stubby, and those in 
the other two solutions did not develop any nodules at all. Their leaves were 
also yellowish and the roots dark. Plate 1 shows the root development 
and nodule formation in the different solutions. 



EFFECT OF REACTION ON NODULE FORMATION 279 

Growth and inoculation of soybeans in Crone's nutrient solution at different 

reactions 

The principal experiment on the influence of reaction on growth and inocu- 
lation of soybeans was next started with Crone's nutrient solution. The 
buffer condition of this solution was, however, unsatisfactory in the alkaline 
range and for this reason several different substances, namely, di-basic sodium 
phosphate, di-basic potassium phosphate, sodium glycerophosphate, and sodium 
carbonate and bicarbonate were added and tested for their buffer effect, and 
also influence on nodule formation. Of these, sodium carbonate at the rate 
of f gm. per liter proved to be the most satisfactory, and hence was used. 
The salts for Crone's solution were ground to a fine powder and thoroughly 
mixed in the following proportions. 

am. 

KC1 100 

CaS0 4 -2H 2 25 

MgS0 4 -7H 2 25 

Ca»(PO«)« 25 

FePO, 25 

For the solution, 12 gm. of this mixture were added to 8 liters of distilled 
water. The mixture was well shaken with the water and left to stand for one 
day, at end of which time the solution was filtered to remove the insoluble 
materials. The acid reactions were obtained by adding varied amounts of 
sulfuric acid. The alkaline reactions were obtained by first adding the sodium 
carbonate and then acid until the desired reaction existed. 

The plants were grown in percolators according to the general method 
already outlined. Good plant growth took place at the favorable reactions. 
In all cases duplicates agreed as to nodule formation and root development. 
Plate 2 serves to indicate the relative size of plants and nodules per plants 
in solution cultures. It will be noted from plates 3 to 10, inclusive, that plants 
at pH 3, 4 and 10 had no nodules. Plants at pH 4.5 which is not given in 
figure 2 developed a few nodules, but at this reaction the roots were dark and 
stubby. Plants at pH 5 had fair inoculation. The tops of the plants at 
this reaction were as tall as those at pH 6, but were yellow and much less 
vigorous. Plants at pH 6 and 7 were decidedly the best, both as to growth and 
inoculation. Those at pH 6 were perhaps a trifle better than those at pH 7. 
Plants at pH 8 had some nodules but the roots were slightly brown. The 
roots at pH 9 and 10 were still browner and did not become inoculated. It 
should be noted that plants at pH 9 were a little taller than at pH 8, but of a 
very poor color. In all other cultures at pH 8 the plants were better than at 
pH 9. Table 3 gives dry weight of plants and number of nodules per plant. 

The experiment just described was repeated using sand cultures according 
to general method already given. The same solution and reactions, were 
used as in the previous experiment. The extent of nodule formation was 
about the same as that in the solution cultures. The limits of nodule forma- 



280 



O. C. BRYAN 



tion were pH 4 and 8. There was less injury from an unfavorable reaction 
at pH 3, 4, 5, 9 and 10 than in the solution cultures at these reactions. This 
was perhaps due to a smaller amount of nutrient solution actually in contact 
with the plant roots in the sand cultures than in the solution cultures, and 
hence a lesser amount of acid or alkali, making it easier for the plants to 
change the actual reaction to a more favorable one. Plate 13 gives the 
comparative growth in sand cultures. 

TABLE 3 
Growth and inoculation of soybeans in Crone's nutrient solution at different reactions 





DRY WEIGHT PER 






REACTION 


PLANT 


PLANT 


PLANT DEVELOPMENT 


pH 


gm. 






3.30 


0.24 





Tops dead 
Roots poor 


3.97 


0.40 





Tops about dead 
Roots poor 


4.95 


1.00 


30 


Tops fair 

Roots dark and stubbed 


6.50 


1.42 


77 


Tops good 
Roots good 


7.40 


1.42 


68 


Tops good 
Roots good 


8.20 


1.15 


21 


Tops good 

Roots slightly brown 


8.70 


1.00 


3 


Tops fair 
Roots brown 


9.60 


0.60 





Tops poor and yellow 
Roots very brown 



Comparative growth of soybeans, com and cowpeas at different reactions 

For a comparison with the soybean, corn and cowpeas were grown in both 
solution and sand cultures with Crone's solution in exactly the same manner 
as the previous experiment with soybeans, except that nitrates instead of 
chlorides were used in the solutions for corn. 

Plates 11, 12 and 14 serve to indicate the general growth which the corn 
and cowpeas made. The corn in the alkaline range of the solution cultures 
did not make good growth and the leaves were yellow. This was perhaps due 
to the lack of iron in solution, which was caused by precipitation under the 
alkaline conditions. The corn in the sand cultures grew better than in solu- 
tion cultures and none of it became yellow. Corn plants at pH 5 in the sand 
cultures were inferior to the other plants at the beginning of the experiment 
and never grew as well as plants at pH 4. The maximum growth of corn 
took place at pH 6 and 7 in the sand cultures, and at pH 5 and 6 in the solu- 
tion cultures. Plate 15 indicates that corn can grow at a considerably more 



EFFECT OF REACTION ON NODULE FORMATION 



281 



acid reaction than the cowpeas and soybeans. Apparently, as indicated in 
table 4 cowpeas have a wider range of inoculation than the soybeans. The 
corn was not grown in duplicate cultures and hence any conclusion with 
corn must be tentative. 

TABLE 4 
Growth and inoculation of cowpeas in sand cultures at different reactions 



REACTION 


NODULES PER PLANT 


PLANT DEVELOPMENT 


pB 






3.3 





Tops fair 

Roots stubby and brown 


3.97 


20 


Tops fair 

Roots slightly dark 


4.95 


22 


Tops and roots good 


6.5 


32 


Good plants, roots, and tops 


7.4 


33 


Good plants, roots, and tops 


8.2 


26 


Tops good 

Roots slightly brown 


8.7 


19 


Tops fair 
Roots brown 


9.6 


14 


Tops fair 

Roots very brown 



The influence of the reaction of the culture medium on the reaction of the plant 

juices 

The influence of the reaction of the culture medium on the reaction of the 
plant juices has been investigated by several workers (17, 38); and it was 
thought desirable to obtain additional information along this line. The reac- 
tion of the juices of the roots and leaves of some of the plants grown in the 
previous experiments was determined electrometrically by the method de- 

TABLE S 
The reaction of the juices of plants grown in media of different reactions 



REACTION OF 


SOYBEANS 


COWPEAS 


CORN 




Leaves 


Roots 


Leaves 


Roots 


Leaves 


Roots 


pH 


pH 


pH 


PH 


pH 


pH 


pn 


3.30 


5.60 


4.68 


5.33 


4.89 


5.19 


4.99 


3.97 


5.90 


5.09 


5.37 


5.37 


5.20 


5.46 


4.95 


6.08 


5.29 


5.38 


5.77 


5.18 


5.55 


6.50 


6.11 


5.61 


5.41 


5.95 


5.21 


5.71 


7.40 


6.12 


5.75 


5.47 


6.07 


5.20 


5.90 


8.20 


6.11 


5.85 


5.50 


6.14 


5.19 


5.90 


8.70 


6.14 


6.29 


5.53 


6.25 


5.21 


6.10 


9.65 


6.16 


7.12 


5.46 


6.58 


5.11 


6.32 



282 



O. C. BRYAN 



scribed by Clevenger (8) and Haas (17). The results are recorded in table 5. 
In general, the reactions of the plant juices as had previously been suggested 
(38) became more acid as the culture medium rose in acidity. The corn tops, 
however, showed no appreciable change. The reactions of the root juices 
were more nearly like those of the culture medium than were those of the 
leaves. 

DISCUSSION 

In making a comparison of the data presented, it should be clearly under- 
stood that pH 3 signifies an acidity ten times greater than pH 4, and pH 4 ten 
times greater than pH 5, etc. Hence, a small change in pH means a great 
difference in hydrogen-ion concentration. It will be noted from the data 
that the most favorable reaction for plant growth and nodule formation with 
soybean was from pH 6 to 7, which is a condition of slight acidity. This fact 

TABLE 6 

Change in reaction of Crone's nutrient solution in contact with soybean roots for 24 hours, 
as determined by the hydrogen electrode 



CULTURE NUMBER 


INITIAL REACTION 


FINAL REACTION 




pH 


PE 


3 


3.30 


3.30 


4 


3.95 


4.00 


5 


4.80 


5.10 


6 


6.90 


6.10 


7 


7.80 


7.00 


8 


8.70 


7.90 


9 


9.50 


8.80 


10 


9.70 


9.40 



was indicated more clearly in the solution cultures than in the sand culture. 
The reactions at which maximum growth took place agrees with those reported 
by Hoagland (23) and Salter and Mcllvane (34). Corn grew at a much 
stronger acidity than soybeans. This conforms with field observations. 

The unsatisfactory results obtained in solution cultures with the modified 
form of Shive's nutrient solution are not readily explained since the plants 
grew well and became inoculated fairly well in the sand cultures. It was 
thought that possibly the salts used contained toxic substances which the 
sand adsorbed and thus prevented their toxic effect on the plants. In order 
to test this possibility some of the solution was thoroughly shaken with 
activated charcoal before being used. This appreciably lessened the injurious 
effects on the roots, but still no nodules appeared. Wilson (39) failed to 
obtain any nodules with soybeans in solution cultures using Pfeffer's nutrient 
solution even after varying the amount of nitrates. This same thing was 
found to be true with Shive's nutrient solution in the present investigation. 



EFFECT OF REACTION ON NODULE FORMATION 283 

German and Didlake (13) reported inoculation of soybeans in solution cultures, 
using a nitrogen free commercial fertilizer for the nutrient salts. The writer 
believes that the unsatisfactory results obtained with Shive's nutrient solution 
in solution cultures were possibly due to toxic impurities in the salts used. 

Crone's nutrient solution was not entirely satisfactory in the alkaline range 
because of the difficulty of maintaining a constant reaction when in contact 
with growing plants. This was even true to some extent after the addition of 
sodium carbonate as is indicated in table 6. The acid range was much more 
constant than the alkaline range, although it changed some. This same 
difficulty exists, of course, to some extent with all nutrient solutions, especially 
if the plants grow well in them. 

The carbon dioxide excreted by the plant roots and that absorbed from the 
atmosphere were no doubt large factors in changing the reaction in the alkaline 
range. 

It was found in the present investigation that when sodium glycero phos- 
phate was added to Crone's solution the reaction remained almost constant 
for a daily period, but no nodules were formed in solution cultures. The 
addition of dibasic potassium phosphate to the alkaline range of Crone's solu- 
tion was also found to hold the reaction of the solution fairly constant for 
daily periods, but nodule formation was again poor. In general, the reaction 
of a solution which was fairly favorable to plant growth so that considerable 
growth took place, would not remain constant very long in contact with the 
growing plants, unless the initial reaction of the solution was exactly the one 
most favorable for the plants. The change in reaction of the solution due 
to growing plants was always in the direction of a more favorable reaction. 
Salter and Mcllvane's data (34) indicate that the reaction of their nutrient 
solution in contact with growing plants remained nearly constant during 
four-day periods. This was possibly due to the slow rate of growth of their 
plants. 

The data show that pure cultures of soybean bacteria are able to grow at an 
acid reaction almost as great as that of the host plant. These results are 
not exactly in line with those of Bewley and Hutchinson (2) who reported that 
definitely acid soils would finally kill the nodule bacteria of lupines, red 
clover, and broad bean. However, they did not state the exact degree of 
acidity of the soil used and hence it is possible that they worked with very 
strongly acid soils. It will be seen from tables 2 and 3 that the best inocula- 
tion took place at a slightly acid to neutral reaction, although some inocula- 
tion took place at a reaction of pH 5 and even pH 4. The information secured 
thus far does not indicate that the critical hydrogen-ion concentration of pure 
cultures of the soybean bacteria is different from what it is in the soil. 

The results show distinctly that the reaction of the media in which the 
soybeans are grown has a direct influence on growth and inoculation. The 
reactions which produced injury and poor inoculation were within the range 
of reactions of actual soil solutions and suspensions as reported by Gillespie 



284 O. C. BRYAN 

(15) Sharp and Hoagland (35), Plummer (32) Truog (37) and others. Differ- 
ent plants vary considerably in their ability to grow at reactions of pH 4, 5, 
and 6. Thus it will be seen that a proper adjustment of the reaction of 
the soil for different plants is of prime importance for the best growth and 
inoculation. 

The reaction of the media in which the plants are grown often has a direct 
influence on the reaction of the plant juices. Further studies on the influence 
of reaction on legumes and legume bacteria are in progress. 

SUMMARY 

A study was made of the influence of acidity and alkalinity on growth 
and inoculation of soybeans in solution and sand cultures. The plants were 
grown in 500- and 600-cc. percolators. A modified form of Shive's nutrient 
solution was used at first, but with unsatisfactory results in solution cultures. 
Three other nutrient solutions were tested for influence on inoculation in 
solution cultures. Of these, Crone's nutrient solution proved to be the most 
satisfactory, and was selected with slight modification for the principal work 
reported herein. The reactions of the solutions were adjusted by adding 
varied amounts of acid or alkali as the case required. The reactions were 
kept as constant as possible by renewing the solutions daily. The old solu- 
tions were removed from the sand with suction before the new solutions were 
added. The cultures were allowed to grow from 25 to 35 days after inocula- 
tion. A few cultures of corn and cowpeas were grown under similar condi- 
tions to that of the soybean for comparison. Twenty-one different strains of 
soybean bacteria were grown on pure cultures of different reactions in order to 
compare the critical hydrogen-ion concentration of the soybean bacteria with 
that of the host plant. 

1. Shive's nutrient solution was favorable for growth and inoculation of 
soybeans in sand cultures, but not in solution cultures. It seems possible 
that there were toxic impurities in the nutrient salts used which were adsorbed 
by the sand. 

2. Crone's nutrient solution was favorable for growth and inoculation in 
both solution and sand cultures. The alkaline range of this solution has a 
very poor buffer action, which is improved markedly by the addition of 
f gm. of sodium carbonate per liter. 

3. The reaction of the nutrient solution in contact with growing plants does 
not remain constant very long, unless the initial reaction of the solution is the 
most favorable one for the plants. The rate of change in reaction is greater 
in the alkaline range than in the acid range. Plants growing rapidly appar- 
ently influence this rate of change more than slow growing plants. 

4. The most favorable reaction for growth and inoculation of soybeans 
was pH 6.5. The limits for which inoculation took place were about pH 4.6 
and 8. The limits for growth of soybeans were about pH 3.9 and 9.6. Reac- 
tions pH 4.95 and 8.2 are injurious to the growth of soybeans, but do not 
entirely prevent inoculation. 



EFFECT OF REACTION ON NODULE FORMATION 285 

5. The hydrogen-ion concentrations which were markedly injurious to 
plant growth and inoculation in this investigation were not any greater and 
in some cases considerably less than the hydrogen-ion concentration of very 
acid soil solutions and suspensions as reported by recent investigators. 

6. The critical hydrogen-ion concentration for nodule formation of soybeans 
was slightly less than that for plant growth. The different strains of 
soybean bacteria showed small differences in regard to critical hydrogen-ion 
concentration. 

7. Corn grew at a greater acidity and alkalinity than the soybean or cowpea. 
The cowpea apparently has a greater range of reaction at which nodules are 
formed than the soybean. 

8. The reaction of plant juices varied with the degree of acidity or alkalinity 
at which the plants were grown, except in the case of the juice of the corn 
leaves which showed little change. The juices of the roots followed the reac- 
tion of the media more closely than did the juices of the leaves. 

REFERENCES 

(1) Abbott, J. B., Conner, S. D. and Smalley, H. R. 1913 Soil acidity, nitrification 

and the toxicity of soluble salts of aluminum. Ind. Agr. Exp. Sta. Bui. 170. 

(2) Bewley, W. F., and Hutchinson, H. B. 1920 On the change through which the 

nodule organism (PS. Radicicola) passes under cultural conditions. In Jour. 
Agr. Sci., v. 10, part 2, p. 144-162. 

(3) Breazeale., J. F., and LeClerc, J. A. 1912 The growth of wheat seedlings as 

affected by acid or alkali conditions. U. S. Dept. Agr. Bur. Chem. Bui. 149. 

(4) Cameron, F. K., and Breazeale, J. F. 1904 The toxic action of acids and salts 

on seedlings. In Jour. Phys. Chem., v. 8, no. 1, p. 1-13. 

(5) Clark, W. M., and Lubs, H. A. 1917 The colorimetric determination of hydrogen- 

ion concentration and its application to bacteriology. In Jour. Bact., v. 2, 
no. 1, p. 109-191, v. 3, p. 217-242. 

(6) Conner, S. D. 1921 Liming and its relation to injurious inorganic compounds in 

the soil. In Jour. Amer. Soc. Agron., v. 13, no. 3, p. 115-124. 

(7) Crone, G. von der 1902 Ergebnisse von Untersuchungen iiber die Wirkung der 

Phosphorsaure auf die hbhere Pflanze. In Sitzber. Gesell. Natur. u. Heilk. Bonn, 
1903, p. 167-173. 

(8) Clevenger, C. B. 1919 Hydrogen-ion concentration of plant juices. In Soil Sci., 

v, 8, no. 3, p. 217-242. 

(9) Duggar, B. M. 1920 Hydrogen-ion concentration and the composition of nutrient 

solutions in relation to the growth of seed plants. In Ann. Mo. Bot. Gar., v. 7, 
no. 1, p. 1-49. 

(10) Dachnowsbdc, A. 1914 The effect of acid and alkaline solutions upon the water 

relation and metabolism of plants. In Amer. Jour. Bot., v. 1, p. 412-443. 

(11) Fred, E. B., and Loomis, N. E. 1917 Influence of hydrogen-ion concentration on 

the development of alfalfa bacteria. In Jour. Bact., v. 2, p. 629-633. 

(12) Fred, E. B., and Davenport, Audrey 1918 Influence of reaction on nitrogen assim- 

ilating bacteria. In Jour. Agr. Res., v. 14, no. 8, p. 317-336. 

(13) Garman, H., and Dedlake, Mary 1914 Six different species of nodule bacteria. 

Ky. Agr. Exp. Sta. Bui., 184. 

(14) Gruzit, O. M. 1917 The effects of some acids and alkalies on soil bacteria in solution. 

In Soil Sci., v. 3, p. 289-295. 



286 0. C. BRYAN 

15) Gillespie, L. J. 1916 The reaction of the soil and measurement of hydrogen-ion 

concentration. In Jour. Wash. Acad. Sci., v. 6, no. 1, p. 7-16. 

16) Gillespie, L. J. 1920 Colorimetric determination of hydrogen-ion concentration 

without buffer mixtures, with special reference to soils. In Soil Sci., v. 9, no. 2, 
p. 115-136. 

17) Haas, A. R. C. 1920 Studies on the reactions of plant juices. In Soil Sci., v. 9, 

no. 5, p. 341-368. 

18) Hartwell, B. L., and Pember, F. R. 1907 The relation between the effect of liming 

and different nutrient solutions containing different amounts of acids, upon the 
growth of certain cereals. In R. I. Agr. Exp. Sta. 20th Ann. Rpt., p. 358-380. 

19) Hartwell, B. L., and Pember, F. R. 1918 The presence of aluminum as a reason 

for the difference in effect of so-called acid soils on barley and rye. In Soil Sci., 
v. 6, p. 259-277. 

20) Heald, F. D. 1896 On the toxic effects of dilute solutions of acid and salts on plants. 
In Bot. Gaz., v. 22, no. 2, p. 125-153. 

21) Hiltner, L. 1915 The sensitiveness of different lupines and other plants to lime. 

Prakt. Bl. Pflanzinbau u. Schutz, n. s., 13, no. 5, p. 53-59; abs. Exp. Sta. Rec, 
v. 35, p. 430. 

22) Hlxon, R. M. 1920 The effects of reaction of a nutrient solution on germination 

and first stages of plant growth. In Meddel. K. Vet. Akad. Nobel-Institute, 
v. 4, no. 9, p. 1-28 

23) Hoagland, D. R. 1918 Effect of hydrogen- and hydroxyl-ion concentration on 
growth of barley seedlings. In Soil Sci., v. 3, no. 6, p. 559-560. 

24) Hopkins, C. G., and Pettit, J. H. 1910 Soil Fertility Laboratory Manual, p. 22 
Ginn & Co., New York. 

25) Joffe, J. S. 1920 The effects of soil reaction on the growth of alfalfa. In Soil Sci., 
v. 10, no. 4, p. 301-307. 

26) Kahlenberg, L., and Trtje, R. O. 1896 On the toxic action of dissolved salts an J 

their electrolytic dissociation. In Bot. Gaz., v. 22, no. 2, p. 81-124. 

27) Kappen, H. 1918 Untersuchangen und Wurzelsaften. In Landw, Vers. Stat., 

Bd. 91, p. 40. 

28) Kappen, H. 1920 tJber die Aziditatsforms des Bodens und ihre Phlanzenphysiolo- 
gische Bedeutung. In Landw. Vers. Stat., v. 96, p. 306-307. 

29) Loew, F. A. 1903 Toxic effects of hydrogen- and hydroxyl-ions on seedlings of 
Indian corn. In Science, n. s., v. 18, no. 453, p. 304-308. 

30) Mirasol, J. J. 1920 Aluminum as a factor in soil acidity. In Soil Sci., v. 10, no. 3, 
p. 153-219. 

31) McCall, A. G. 1916 Physiological balance of nutrient solution for plants in sand 
cultures. In Soil Sci., v. 2, p. 207-254. 

32) Plummer, J. K. 1918 Studies on the soil reaction as indicated by the hydrogen 
electrode. In Jour. Agr. Res., v. 12, p. 19-31. 

33) Salter, R. C. 1916 The behavior of legume bacteria on acid and alkali media. In 

Proc. Iowa Acad. Sci., v. 23, p. 309-313. 

34) Salter, R. M., and McIlvane, F. C. 1920 The effect of reaction of solution on 

germination of seed and growth of seedlings. In Jour. Agr. Res., v. 19, no. 2, 
p. 73-96. 

35) Sharp, L. T., .and Hoagland, D. R. 1916 Acidity and absorption of the soil as 

measured by the hydrogen electrode. In Jour. Agr. Res., v. 7, no. 3, p. 123-145. 

36) Shive, J. W. 1918 A study of physiological balance in nutrient media. In Physiol. 
Res., v. l,no. 7, p. 327-397. 

37) Truog, E. 1918 Soil acidity: Its relation to the growth of plants. In Soil Sci. 
v. 5, no. 3, p. 169-195. 



EFFECT OF REACTION ON NODULE FORMATION 



287 



(38) Truog, E., and Meacham, M. R. 1919 Soil acidity: Its relation to the acidity of 

plant juices. In Soil Sci., v. 7, no. 6, p. 469-475. 

(39) Wilson, J. K. 1917 Physiological studies of the Bacillus Radkicola of the soybean 

(Soja Max Piper) and of the factors influencing nodule production. N. Y. 
(Cornell) U. Agr. Exp. Sta. Bui. 285. 



SOIL SCIENCE, VOL. XIII, NO. 4 




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Soybean Roots Grown in Solution Cultures with pH Values of Approximately 

3 and 4 



290 



EFFECT OF REACTION ON NODULE FORMATION 

O. C. BRYAN 



PLATE 4 




Soybean Roots Grown in Solution Culture with pH Value of Approximately 4.5 

291 



EFFECT OF REACTION ON NODULE FORMATION 

O. C. BRYAN 



PLATE 5 







Soybean Roots Grown in Solution Culture with pH Value of Approximately 5 



292 



EFFECT OF REACTION ON NODULE FORMATION 

O. C. BRYAN 



PLATE 6 




Soybean Roots Grown in Solution Culture with pH Value of Approximately 6 



293 



EFFECT OF REACTION ON NODULE FORMATION 

O. C. BR tf AN 



PLATE 7 




Soybean Roots Grown in Solution Culture with pH Value of Approximately 7 



294 



EFFECT OF REACTION ON NODULE FORMATION 



PLATE 8 



O. C. BRYAN 




Soybean Roots Grown in Solution Culture with pH Value of Approximately 8 



295 



EFFECT OF REACTION ON NODULE FORMATION 

O. C. BRYAN 



PLATE 9 




Soybean Roots Grown in Solution Culture with pH Value of Approximately 9 



296 



EFFECT OF REACTION ON NODULE FORMATION 

O. C. BRYAN 



PLATE 10 




Soybean Roots Grown in Solution Culture with pH Value of Approximately 10 



297 




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EFFECT OF REACTION ON NODULE FORMATION 

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PLATE 15 




Corn, Soybeans and Cowpeas Grown in Solution Cultures of Approximately pH 4 . 

in each Case 



302 



LIBRARY OF CONGRESS 



000 930 452 6 



SOIL SCIENCE 

VOLUME 13, NUMBER 4, APRIL, 1922 



CONTENTS 



C. T. Hirst and J. E. Greaves. Factors Influencing the Determination of Sulfates in Soil.. 231 
J. E. Greaves and E. G. Carter. The Influence of Moisture and Soluble Salts on the 

Bacterial Activities of the Soil 251 

O. C. Bryan. Effect of Different Reactions on the Growth and Nodule Formation of Soybeans. 271 
William M. Gibbs and C. H. Werkman. Effect of Tree Products on Bacteriological Activi- 
ties in Soil: I. Ammonification and Nitrification 303 



